Tumors larger than microscopic size have inadequate and abnormal vasculature. As a consequence, areas of tumors that are more than a few tens of microns from a capillary are generally hypoxic. Cells in such hypoxic areas of tumors either die or convert to anaerobic metabolism which results in their excreting lactic acid. Because of the poor circulation in tumors, that excreted lactic acid builds up in the interstitial space in hypoxic areas of tumors.
There are two important consequences of this process.
1) While the pH in the interstitial space in normal tissues ranges from about 7.2 to 7.5, the interstitial space in hypoxic areas of tumors is acidic, with pH ranging from as low as about 6.0 in areas most distant from capillaries, up to about 7.0 closer to capillaries.2) While tumor cells in close proximity to capillaries are characterized by high metabolic rates and fast cell division, the tumor cells in acidic areas more distant from capillaries have lower metabolic rates and are slower-dividing or non-dividing. The slow-dividing and non-dividing tumor cells are referred to as quiescent.
Conventional cancer therapies, including chemotherapy and radiation, are generally fairly effective in killing fast-dividing cells, but because conventional cancer therapies are explicitly selected on the basis of their ability to spare slow-dividing and non-dividing cells typical of most normal tissues, said cancer therapies are also relatively ineffective against slow-dividing and non-dividing quiescent tumor cells. As a consequence, cancer treatments typically kill predominantly the fast-dividing cells of a tumor, while sparing the quiescent cells of the tumor. The initial killing of the fast-dividing tumor cells causes the tumor to go into remission. After those killed cells have been disposed of by the body's normal cleanup processes, all too often the treatment-resistant quiescent cancer cells in the hypoxic areas of the tumor slowly regain access to adequate oxygen, nutrients, and waste disposal, thus allowing them to revert to high metabolic rate and fast cell division. Reversion of the previously-quiescent cancer cells manifests as the dreaded relapse that so often kills cancer patients.
To achieve more effective treatment of tumors it is desirable to have a means for sensitive detection of quiescent cancer cells in virtually all tumors. Also desired is to have a broadly effective means for selectively killing those treatment-resistant quiescent cancer cells without concomitant killing of cells in normal tissues.
1. Related Art
There are two distinct types of compositions for detecting and treating cells in acidic areas of tumors: embedder compositions and transporter compositions. Compositions of the embedder type position their cargos on the outer surface of cells, as illustrated in Comparative FIG. 1a [RELATED ART]. In contrast, compositions of the transporter type of the instant invention carry their cargos into the cytosol of cells, as illustrated in FIG. 1.
Embedder compositions are described in a co-submitted and co-pending patent application by applicant cited supra and relating to embedder compositions and methods. Said embedder compositions repel from cells at the pH in normal tissues, but embed into membranes and thereby position their cargos on the outer surface of cells in acidic areas of tumors. Embedder compositions contain cargos which are effective on external cell surfaces. Such cargos can be relatively large and/or polar because it is not necessary to pull them across the cell membrane. One important difference between embedder compositions of said co-pending patent application and the transporter compositions of the instant invention is that for therapeutic application the cargo of the embedder composition can be designed to exploit a component of the body's natural extracellular cell-killing machinery, such as phagocytic cells or the innate immune system.
2. Prior Art
In the mid-1990s applicant pioneered the development of transporter peptides designed to transport substances from a low-pH environment across a lipid layer to a higher-pH aqueous compartment. The original transporter peptides are described and claimed in U.S. Pat. No. 6,030,941, issued to Summerton and Weller and assigned to AVI BioPharma, Inc. When 941 was filed in 1997 it was believed by applicant that structures with the highest practical pH of transition (pT) had been devised and were claimed in 941. Those high-pT peptides comprise peptides containing either of the two core repeating sequences [LELLE]n or [ELLLE]n. Furthermore, results from early octanol/buffer partitioning studies suggested that those high-pT peptides should be adequate for delivering substances into virtually all cells in acidic areas of tumors.
However, in subsequent studies of entry of transporter peptides into mammalian cell membranes and into mammalian cells at 37 degrees C., applicant discovered that the high-pT peptides claimed in 941 only enter cell membranes at a substantially lower pH than would be expected on the basis of the early partitioning results. The implication of this new finding is that the early high-pT peptides claimed in 941 will likely be effective only in the most acidic areas of tumors most distant from capillaries. This leaves a substantial portion of the acidic areas of tumors undetected or untreated by transporter compositions utilizing such prior art peptide sequences. This is illustrated in Comparative FIG. 2a [PRIOR ART].
The discovery of the limitations of the high-pT transporter peptides claimed in 941 led applicant to search for transporter peptide sequences having even higher pT values, which it was hoped would be effective to enter cells in a substantially wider range of acidic areas of tumors. Those efforts led to the discovery by applicant of a number of improved transporter peptide sequences patentably distinct from the claims of 941. The improved transporter peptide sequences disclosed and claimed herein have been found to enter mammalian cells at appreciably higher pH values and so should allow one to effectively detect or treat a much larger portion of the acidic areas of tumors, as illustrated in FIG. 2.
Table 1 lists key differences between the prior art transporter peptides described and claimed in 941 and the newly devised improved transporter peptides of the instant invention. These properties will be further described in the detailed description of the invention.
TABLE 1PropertyPrior Art (941)Current Inventionacid side chain content in core40% or moreless than 40%peptide sequence:acid side chains selected from:glutamic and asparticglutamicaverage of axial rotations500 degrees or lessmore than 500between pairs:degrees
Table 2 illustrates these differences for representative core transporter peptide sequences.
TABLE 2PropertyPrior Art (941)Current Inventioncore repeating sequence[LELLE]n (40% E)[ELLELELLLEL]n(36% E)axial rotation between pairs500 degrees550 degreescore repeating sequence[ELLLE]n (40% E)[ELLLELELLEL]n(36% E)axial rotation between pairs500 degrees550 degreescore repeating sequence[ELLLEL]n (33% E)axial rotation between pairs600 degrees
While these differences between the prior art (941) and the current invention may appear modest, because of the typical pH distribution in tumors the improved peptides of the current invention are expected to afford a substantial increase in diagnostic sensitivity, and a dramatic improvement in therapeutic efficacy (when used in combination with conventional cancer therapy to kill the fast-dividing cells of the tumor), as illustrated in Comparative FIG. 2a [PRIOR ART] and FIG. 2.